Abstract
Abstract: :
Purpose: Mutations in FOXC1, a member of the forkhead family, are responsible for Axenfield Rieger malformations and lead to an increased susceptibility to glaucoma. Genes regulated by FOXC1 in normal development and adversely affected by FOXC1 mutations could be involved in glaucoma pathogenesis. Thus, identification of FOXC1 target genes is necessary to understand the molecular steps leading to Axenfeld Rieger malformation. Methods: We used a modified ChIP technique to enrich FOXC1 target genes recovered from fixed complexes of chromatin–His–fusion–proteins using Ni++ columns. Among the specific targets obtained, a 5’upstream element of 1.3kb belonging to FGF19 gene was identified and cloned, as a whole insert or as serial deletions, into the pGL3 luciferase reporter for transactivation and EMSA assays. A putative FOXC1 binding site within the FGF–19 regulatory region was localized and subjected to mutational analysis. Immunohistochemistry and tissue culture of porcine corneal eyes are underway to localize FGF–19 and its receptor FGFR4. Results: FGF–19, a novel member of the fibroblast growth factor gene family, was identified as a candidate FOXC1 target gene and selected for further analysis. FOXC1 was able to transactivate luciferase reporter from the FGF–19 5’–upstream region. Moreover, electromobility shift assays revealed that FOXC1 could specifically bind an element within a 120 bp fragment of the FGF19 regulatory region. Mutagenic abolition of the putative site prevents FOXC1 transactivity from the reporter gene. FGF–19 has been shown to be involved in variety of biological processes including embryonic development cell growth, morphogenesis, tissue repair, tumor growth and invasion. The FGF–19 growth factor is a high affinity, heparin dependent ligand for FGFR4 receptor. The role that FGF–19 may play in the adult eye is not known. However, preliminary data using western blotting showed high FGF–19 expression in the aqueous humour and localization of the FGFR–4 receptor within the anterior segment. Conclusions:These experiments identify FGF–19 as a candidate gene regulated by FOXC1 in the adult eye. Experiments are underway to localize the FGF–19 and its receptor FGFR–4 within the eye and detect the transduction signaling pathways involving FGF–19. In addition to identify a key step toward understanding the mechanisms underlying FOXC1 regulation, this data may assist in the development of efficient treatments for Axenfield Rieger malformation.
Keywords: anterior segment • transcription factors • growth factors/growth factor receptors